1. Field of the Invention
This invention relates to a rapid dot immunobinding assay. More particularly, the invention relates to a dot immunoassay in which the antigen or antibody in a clearly defined spot is bound onto a hydrophobic membrane which can be secured to a test strip.
2. Description of the Prior Art
Enzyme immunoassays (EIA) are commonly employed for the rapid diagnosis of infectious and other diseases by both antigen and antibody detection. Solid-phase formats include microtiter plate and membrane-based assays (Yolken, R. H., Yale J. Biol. Med.. 53:85-92 (1980). Successful tests have been developed for viruses (Hildreth, S. W., and Beaty, B. J., Am. J. Trop. Med. Hyg., 33:965-972 (1984); Scott, T. W. and Olson, J. G., Am. J. Trop. Med. Hyg., 35:611-618 (1986); Tsai, et al., J. Clin. Microbiology. 25:370-376 (1987) as well as bacteria and other agents (Sippel et al., J. Clin. Microbiol., 20:259-265 (1984); Beutin, et al., J. Clin. Microbiol., 19:371-375 (1984); Williams et al., J. Clin. Microbiol., 24:929-934 (1986); Smith, S. and C. F. Repetti, J. Clin. Microbiol. 25:2207-2208 (1987). Sensitivity and specificity of EIA has been shown to be comparable to fluorescent antibody (FA) (Hildreth, S. W., and Beaty, B. J., Am. J. Trop. Med. Hyg., 33:965-972 (1984)), radioimmunoassay (RIA) (Voller et al., Manual of Clinical Immunology, American Society for Microbiology, Washington, D.C. (1976)) and hemagglutination inhibition (HI) tests (Calisher et al., J. Clin. Microbiol., 24:770-774 (1986)).
In existing membrane based immunoassays, antigens and antibodies are bound to the membrane in a variety of ways, including gravity filtration and vacuum filtration. These methods as applied to hydrophobic membranes do not result in a clearly defined spot and efficient binding. Attempted solutions to this problem include modifying the hydrophobic membrane to make it hydrophilic. As a consequence, the sensitivity and practicality of the methods is not optimal. There is a need for an improved method in which binding of the antigen or antibody to a membrane gives a clearly defined spot of uniform density.
There is no mention, in the literature, of the improvement in signal-to-noise ratio by inhibiting the interaction of the test solution and the membrane. Hydrophilic membranes freely interact with test solutions and any materials, molecules, etc., contained in that solution. This causes non-specific background. Using hydrophobic membranes and achieving a balance between surface action and hydrophobicity limits undesirable backgrounds. Where hydrophobic membranes have been used before, the membrane has been wetted by solvent or surfactant prior to use.
Many viruses, such as eastern equine encephalitis (EEE) and St. Louis Encephalitis (SLE) as well as many bacteria, are significant public health threats. EEE virus infects humans and horses, frequently causing acute disease with a high rate of mortality. SLE virus is a major cause of viral encephalitis in humans. One means of combatting epidemics of these diseases is to identify a given area of high risk and subsequently control the vectors. Assaying the sera from sentinel flocks of chickens, for example, has shown to be an effective and sensitive method for arbovirus surveillance. The enzyme immunoassay (EIA) is a promising alternative to traditional assays for screening of sentinel animal sera. The technique does, however, require trained personnel to perform and read the test and specific equipment, and may take several hours to complete. For these reasons, an improved EIA is needed for field use.